Abstract: :
Purpose: In the retina, Müller glial cells play a pivotalrole in the modulation of synaptic transmission and expressfunctional N–methyl–D–aspartate (NMDA) glutamatereceptors, which may regulate glial activity by establishingfeedback loops between glia and neurons. Glycine acts as a co–agonistat these receptors through interactions with strychnine–insensitivesites. N–methyl–D–aspartate receptors (NMDARs)differ in the retina and the brain regarding properties of glycinecoagonist–binding site, which confer distinct functionalcharacteristics to the receptor. We present the pharmacologicalcharacterization of the glycine binding site and the molecularanalysis of the subunit expression as well as the alternativesplicing–generated NR1 isoform composition of NMDA receptorsin Müller cells.Methods: Specific binding of radiolabeled ligands in the presenceof cold competitors to membrane fractions from Müller gliaand retinal neuron primary cultures was determined. Radioligandbinding data were analyzed via nonlinear regression using PRISMcomputer program. The molecular composition of NMDA receptorwas analyzed by RT–PCR and Western blot.Results: Glycine binds specifically to NMDA receptors in thesecells in a saturable, pH–dependent, temperature–independentfashion. Importantly, we report cell–specific differencesin properties of the strychnine–insensitive glycine bindingsite and NR1 splice variant expression in neurons and Müllerglia. Glycine–binding to cultured Müller cells showslower affinity (Kd=632±198 nM) and higher density (Bmax=43.58±7.02pmol/mg protein) than in neurons. D–serine, postulatedas the endogenous glutamate coagonist at NMDARs competitivelyinhibits glycine binding with up to 60 fold lower affinity thanglycine. Spermine has no effect on the glial coagonist site,in contrast with the stimulation by this polyamine of neuronalglycine–binding and NMDAR activity. Molecular analysisof NR2 subunit expression and of N–terminal and C–terminalNR1 variants in MC further confirms NMDAR structural differencesin neurons and glia which could underlie a differential regulationof glutamate function in the retina.Conclusions: The observed structural differences between glialand neuronal cells in the retina could account for cell–specificpharmacological characteristics of the receptor and underliethe physiological differences between both cell types. Furtherunderstanding of these differences could shed a light on themechanisms through which glia integrates neuronal inputs andparticipates in the modulation of synaptic activity.